It is well known that most of the bodily states in mammals, including most disease states, are effected by proteins. Proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man.
Classical therapeutics generally has focused upon interactions with proteins in an effort to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the production of proteins by interactions with the molecules (i.e., intracellular RNA) that direct their synthesis. These interactions have involved hybridization of complementary "antisense" oligonucleotides or certain analogs thereof to RNA. Hybridization is the sequence-specific hydrogen bonding of oligonucleotides or oligonucleotide analogs to RNA or to single stranded DNA. By interfering with the production of proteins, it has been hoped to effect therapeutic results with maximum effect and minimal side effects.
The pharmacological activity of antisense oligonucleotides and oligonucleotide analogs, like other therapeutics, depends on a number of factors that influence the effective concentration of these agents at specific intracellular targets. One important factor for oligonucleotides is the stability of the species in the presence of nucleases. It is unlikely that unmodified oligonucleotides will be useful therapeutic agents because they are rapidly degraded by nucleases. Modification of oligonucleotides to render them resistant to nucleases therefore is greatly desired.
Modification of oligonucleotides to enhance nuclease resistance generally has taken place on the phosphorus atom of the sugar-phosphate backbone. Phosphorothioates, methyl phosphonates, phosphoramidates and phosphorotriesters have been reported to confer various levels of nuclease resistance. Phosphate-modified oligonucleotides, however, generally have suffered from inferior hybridization properties. See, e.g., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications (CRC Press, Inc., Boca Raton Fla., 1993).
Another key factor is the ability of antisense compounds to traverse the plasma membrane of specific cells involved in the disease process. Cellular membranes consist of lipid-protein bilayers that are freely permeable to small, nonionic, lipophilic compounds and are inherently impermeable to most natural metabolites and therapeutic agents. See, e.g., Wilson, Ann. Rev. Biochem. 1978, 47, 933. The biological and antiviral effects of natural and modified oligonucleotides in cultured mammalian cells have been well documented. It appears that these agents can penetrate membranes to reach their intracellular targets. Uptake of antisense compounds into a variety of mammalian cells, including HL-60, Syrian Hamster fibroblast, U937, L929, CV-1 and ATH8 cells has been studied using natural oligonucleotides and certain nuclease resistant analogs, such as alkyl triesters and methyl phosphonates. See, e.g., Miller, et al., Biochemistry 1977, 16, 1988; Marcus-Sekura, et al., Nuc. Acids Res. 1987, 15, 5749; and Loke, et al., Top. Microbiol. Immunol. 1988, 141, 282.
Often, modified oligonucleotides and oligonucleotide analogs are internalized less readily than their natural counterparts. As a result, the activity of many previously available antisense oligonucleotides has not been sufficient for practical therapeutic, research or diagnostic purposes. Two other serious deficiencies of prior art compounds designed for antisense therapeutics are inferior hybridization to intracellular RNA and the lack of a defined chemical or enzyme-mediated event to terminate essential RNA functions.
Modifications to enhance the effectiveness of the antisense oligonucleotides and overcome these problems have taken many forms. These modifications include base ring modifications, sugar moiety modifications and sugar-phosphate backbone modifications. Prior sugar-phosphate backbone modifications, particularly on the phosphorus atom, have effected various levels of resistance to nucleases. However, while the ability of an antisense oligonucleotide to bind to specific DNA or RNA with fidelity is fundamental to antisense methodology, modified phosphorus oligonucleotides have generally suffered from inferior hybridization properties.
Replacement of the phosphorus atom has been an alternative approach in attempting to avoid the problems associated with modification on the pro-chiral phosphate moiety. For example, Matteucci, Tetrahedron Letters 1990, 31, 2385 disclosed the replacement of the phosphorus atom with a methylene group. However, this replacement yielded low affinity compounds with nonuniform insertion of formacetal linkages throughout their backbones. Cormier, et al., Nucleic Acids Research 1988, 16, 4583, disclosed replacement of phosphorus with a diisopropylsilyl moiety to yield homopolymers having poor solubility and hybridization properties. Stirchak, et al., Journal of Organic Chemistry 1987, 52, 4202, disclosed that replacement of phosphorus linkages by short homopolymers containing carbamate or morpholino linkages to yield compounds having poor solubility and hybridization properties. Mazur, et al., Tetrahedron 1984, 40, 3949, disclosed replacement of a phosphorus linkage with a phosphonic linkage yielded only a homotrimer molecule. Goodchild, Bioconjugate Chemistry 1990, 1, 165, disclosed ester linkages that are enzymatically degraded by esterases and, therefore, are not suitable for antisense applications.
The limitations of available methods for modification of the phosphorus backbone have led to a continuing and long felt need for other modifications which provide resistance to nucleases and satisfactory hybridization properties for antisense oligonucleotide diagnostics and therapeutics.
Consequently, there is considerable interest in developing oligonucleotide surrogates that are capable of maintaining Watson-Crick base pairing to native RNA (DNA) or duplex DNA targets (formation of a triplex) but do not contain the usual phosphodiester linkages. One of the approaches to this problem involves the use of backbones containing peptide type linkages which connect the bases required for base pairing. These compounds are commonly known as peptide nucleic acids (PNA). Thus, in principle, it should be possible to design reagents or molecules that recognize any predetermined sequence, simply by connecting nucleosidic bases or other ligands with appropriate linear spacer molecules to maintain a desired geometry required for recognition of the hydrogen bonding groups in the minor or major groove of a nucleic acid. Recently, PNA and related molecules have demonstrated high affinity and specificity towards nucleic acid targets (Egholm, et. al., J. Am. Chem. Soc. 1992, 114, 1895, 9667; Nielsen, et. al., Science 1991, 254, 1497; Hyrup, et. al., J. Chem. Soc. Chem. Comm. 1993, 518).
Molecular recognition plays a key role in the binding step, that is, the formation of the stable reactive complex. Much work has been done on the so called molecular clefts, where molecules are constructed so that they can recognize specific nucleobases by base pairing or base stacking (Rebek, Jr. Acc. Chem. Res. 1990, 23, 399; Inouye, et. al., J. Am. Chem. Soc. 1992, 114, 778.) It is believed that convergent functionality would provide an advantage in that activity could be `focused` in a highly localized manner at an active site. The ultimate goal is to merge the recognition and reaction steps in space and in time such that maximum binding would occur to transition states, as was anticipated by Pauling, Chem. Eng. News 1946, 24, 1375.
There remains a need in the art for molecules which have fixed preorganized geometry that matches that of a target such as a nucleic acid or protein. The backbone of such molecules should be rigid with some flexibility and easy to construct in solution or via automated synthesis on solid support.